IFN-&#945;subtypes display different biological activities that associate with their binding affinities for the IFN-alpha receptor 1 (IFNAR1) and 2 (IFNAR2). Based on a low energy model, we used the Rosetta program to design a mutant IFN which contains a single amino acid change at position 163 from serine to glutamic acid. This purified IFN mutant protein showed decreased antiviral and antiproliferative activities, suggesting 163 serine is important for IFN-alpha biological functions. We are going to design new IFN-alpha constructs taking into consideration the binding affinity for both IFNAR2 and IFNAR1. We also focused on 14 natural interferon alpha subtypes (IFN-alpha 1a, 2b, 2c, 4a, 5, 6, 7a, 8b, 10a, 14c, 16, 17b, 21a, and 21b). Based on the interferon sequences published, 14 interferon subtype constructs were cloned into a yeast expression vector pKLAC2 and then transformed into a host strainYap3 in which the YAP3 gene encoding yeast aspartic protease 3 (YAP3) was disrupted. Secreted IFN-alpha proteins were collected from culture supernatants and further purified using reverse phase HPLC. Preliminary data showed that the various IFN-alphas exhibit a wide breadth and varied profile of biological activities, suggesting they may play distinct biological roles in living organisms. Further characterization of purified interferon subtype protein is ongoing, including determination of the expression patterns induced by different viruses, the production regulated by different IFN regulatory factor and the signal pathway used. This information will assist in the synthesis of other novel genetically engineered interferons.